Project Summary/Abstract Adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) is a promising therapeutic option for cancer patients, demonstrating a 30-55% objective response rate in clinical trials with metastatic melanoma patients. Current strategies for TIL production are CD8+ T cell-centric, despite indications that CD4+ T cells are prevalent across multiple malignancies, are polyfunctional in nature, and provide therapeutic benefit in a multitude of clinical and pre-clinical applications. The overall objective of this proposal is to systematically investigate the role of CD4+ TIL in the settings of ACT and immunotherapeutic resistance. We hypothesize that CD4+ TIL are instrumental to the effectiveness of ACT and provide an effective mechanism to overcome resistance to current immunotherapy strategies. To investigate this hypothesis, we will 1) Determine the function and phenotype of CD4+ TIL from human melanoma samples, 2) Elucidate the mechanisms of the CD4+ TIL anti-tumor response in vivo, and 3) Examine the contribution of CD4+ TIL in overcoming resistance to immunotherapy. In Aim 1, this study will determine and validate the intrinsic determinants of CD4+ TIL that provide clinical efficacy through the use of in vitro immunologic assays and bioinformatics analysis based on de-identified samples from patients who received ACT with TIL. Aim 2 will approach the mechanism underlying in vivo efficacy through the use of mouse models to address unanswered questions of antigen specificity and antigen recognition by CD4+ T cells. Finally, Aim 3 will utilize complex murine and human systems, including CRISPR/Cas9, to investigate the use of CD4+ TIL to rescue resistance to immunotherapy. The results of this study will provide rationale for the inclusion of CD4+ T cells in TIL products for ACT, especially in the setting of immunotherapeutic resistance. Through the completion of these aims, MacLean will undergo an intensive training in advanced immunology and fundamental principles in bioinformatics and molecular biology that will prepare him for a successful career as an independent investigator. This training is fully supported by his mentor, Dr. Shari Pilon-Thomas, collaborator, Dr. Amod Sarnaik, mentorship committee and the institution, Moffitt Cancer Center and Research Institute. The objective of this training program is to foster the career development of MacLean in basic and translational research through mentorship in 1) tumor immunology and murine models, 2) data analysis and interpretation, 3) oral presentation skills, 4) scientific writing skills, and 5) participation in scientific meetings. Drs. Pilon-Thomas and Sarnaik will meet with MacLean weekly to provide guidance on experimental design and to discuss data analysis and interpretation. MacLean will also meet with members of his mentorship committee on a quarterly basis for valuable added insight into the progress of his project. MacLean will actively participate in advanced courses in immunology, attend national conferences and grant writing workshops, and prepare manuscripts with the overall goal of transitioning from Ph.D. student to postdoctoral fellow and ultimately an independent investigator in translational academic research.